Method for determining precorneal retention time of ophthalmic formulations

ABSTRACT

Apparatus and method for measuring ophthalmic formulation retention on the surface of an eye include dissolving a fluorescent macromolecule in an ophthalmic formulation to form a fluorescently labelled formulation; topically administering the fluorescently labelled formulation to an eye, said fluorescently labelled formulation forming a thin film on the eye surface. Apparatus is provided for illuminating the eye to cause fluorescence of the fluorescently labelled formulation as the fluorescently labelled formulation thin film is eliminated from the eye by normal blinking and lacrimation; and for measuring fluorescence from the thin film as a function of time.

This application is a division of application Ser. No. 07/759,530, filedSep. 13, 1991, now U.S. Pat. No. 5,323,775.

The present invention generally relates to slit lamps for eyeexamination and more particularly, the present invention is directed toa specially modified slit lamp and the use thereof for continuouslymonitoring the emitted fluorescence in topically administered ophthalmicformulations.

Measurement of the retention time of ophthalmic formulations in the eyeis important in the optimization of new ophthalmic dosage forms anddroppable gel systems containing pharmaceutical agents for treatment ofmany eye conditions in addition to contact lens wetting, wound healingand sustained drug release.

Heretofore one of the most common methods utilized in making retentiontime measurements includes the use of sodium fluorescein which is addedto an ophthalmic formulation and instilled into the cul-de-sac of ananimal or human subject to form a thin film over the surface of the eye.The fluorescence signal is then monitored with time using a slit lampfluorophotometer. Unfortunately, this sodium fluorescein is rapidlyeliminated from the film by two predominant mechanisms: It is washed outof the eye by new lacrimal secretions or, being a relatively smallmolecule, it diffuses into the ocular tissue and is thereafter absorbedinto the systemic circulation. The second type of elimination of thesodium fluorescein from the thin film is a source of error in retentiontime measurements as it is difficult to distinguish between fluorescenceof the thin film from fluorescence from the tissue.

In an effort to prevent fluorescein diffusion, attempts have been madeto increase the viscosity of the instilled optical formulations causingthe fluorescein to be retained longer in the precorneal tear film (PTF).(See Benedetto, D. A.; Shah, D. O.; and Kaufman, H. E., "The instilledfluid dynamics and surface chemistry of polymer in the preocular tearfilm," Investigative Ophthalmology, December 1975.) While the referencedarticle shows that fluorescein retention increases by a moderate amountwith increased viscosity of the vehicle, diffusion of fluorescein intothe ocular tissue actually plays a larger role in the elimination offluorescein from the PTF as the retention time of the vehicle increases.Also, it has been reported by the Benedetto et al. reference that thereis little understanding of the relative importance of the role eachphysicochemical property of the polymer plays on the retentionproperties of the polymer solutions, and it is probable that in eachcase different properties assume major importance in determining theoverall retention times.

Therefore, it is not practical to add to proposed or existing eyeformulations viscosity builders in order to drastically reducefluorescein diffusion when eye formulation retention time is to bemeasured and evaluated. It is also well-known that increasing theviscosity of eye formulation can result in eye irritation and/or blurredvision. Hence, subjects typically react by forceful blinking, squeezingthe formulation out of the conjunctival sac, and accelerating thedrainage of the fluorescein tracer.

This is particularly true in comparison testing of eye formulationssince, as reported, added viscosity builders would not uniformly enhancethe properties of the eye formulations, thus making comparative testingnot very reliable.

It is important to realize that with or without such viscosity builders,the diffusion rate of fluorescein out of the PTF severely limits theaccuracy of retention time measurement.

Because there is no bonding between fluorescein and polymers, thefluorescein diffuses freely in the viscous vehicle (Ludwig, A; Unler,N., and VanOoteghem, M., "Evaluation of viscous ophthalmic vehiclescontaining carbomer by slit-lamp fluorophotometry in humans,"International Journal of Pharmaceutics, pp. 15-25, Vol. 61, 1990. Andwhile the carbomer of specific investigation in the Ludwig articleincreased the ocular retention of sodium fluorescein, the retention timewas less than ten minutes.

Other methods for measuring the retention times of ophthalmicformulations in the eye include gamma scintigraphy. However, thesemethods involve the use of radioisotopes and therefore necessitateexpensive equipment and a laboratory suited for the handling ofradioactive compounds. Because of the specialized equipment needed toconduct such testing, it is not cost-effective for a typical laboratoryto make measurements on their ocular formulations. Also, typically, theradioactive compounds have low molecular weights so they too may freelydiffuse out of the viscous vehicle and into ocular tissue or bedeposited on the lid margins that will result in erroneous retentionmeasurements.

Hence there is a need for an apparatus and method suitable for use byindependent laboratories in making important. quantitative measurementsin their development of new ophthalmic dosage forms and formulations.The apparatus and method of the present invention are suited for suchapplication and in fact the optical detection apparatus of the presentinvention can be easily mounted or dismounted from existing slit lamps.This has a distinct advantage for clinicians who wish to evaluate theperformance of a specific ophthalmic formulation for a particularpatient with disease. Because limited additional equipment is requiredto make the measurements, the incremental cost to clinical practice isminimal.

SUMMARY OF THE INVENTION

Apparatus for the measurement of ophthalmic formulation retention on thesurface of the eye, in accordance with the present invention, generallyincludes means for supporting the subject and enabling access of lightto the subject eye, along with means for illuminating the subject eye.Importantly, the means for illuminating the subject eye comprises filtermeans for causing illumination of the subject eye with light of aselected frequency range in order to cause fluorescence of a fluorescentformulation topically administered to the surface of the subject's eyeand forming a thin film thereover. As will be hereinafter described ingreater detail, the filter means may be installed in a conventional slitlamp, thereby enabling the apparatus of the present invention to befabricated from a conventional slit lamp system, thus reducing theoverall cost of the apparatus.

In addition, the apparatus of the present invention includes means formeasuring the fluorescent light from the thin film with such meansincluding filter means for preventing the measurement of backgroundlight signals that can cause "noisy" fluorescence signals.

A method in accordance with the present invention for measuringophthalmic formulation retention on the surface of an eye generallyincludes the steps of dissolving a fluorescent macromolecule in anophthalmic formulation to form a fluorescently labelled formulation andthereafter topically administering the fluorescently labelledformulation to an eye, whereupon the fluorescently labelled formulationforms a thin precorneal film on the eye surface.

Thereafter, the eye is illuminated to cause fluorescence of thefluorescently labelled formulation as the fluorescently labelledformulation thin film is eliminated from the eye by normal blinking andlacrimation. During this time, the fluorescence from the thin film ismeasured.

More particularly, the method in accordance with the present inventionmay include the step of dissolving a fluorescent macromolecule in theophthalmic formulation, the fluorescent macromolecule havingsufficiently large molecular weight to prevent diffusion of the same outof the thin film during illumination of the eye and thereby insuringmeasurement of fluorescence from the precorneal thin film only. Further,the fluorescent molecule may have a molecular weight greater than about2,000 Daltons in order to prevent diffusion of the fluorescentmacromolecule out of the thin film. Diffusion into the ocular tissue isprevented by the large size of the fluorescent macromolecule, andpreferential diffusion of the macromolecule out of the thin film isprevented by entanglements between the macromolecule and the largemolecular weight excipients in the formulation.

In one embodiment of the present invention, the method further includesthe step of covalently attaching a fluorescent compound having amolecular weight of less than about 2,000 Daltons to a compound to forma fluorescent macromolecule.

In combination, the present invention also encompasses a system formeasuring the retention of ophthalmic formulations on the'surface of aneye which includes at least one fluorescently labelled ophthalmicformulation containing a fluorescent macromolecule having molecularweight greater than about 2,000 Daltons, means for illuminating the eyeand causing fluorescence of the fluorescently labelled ophthalmicformulation when the same is topically administered to an eye, and meansfor measuring the fluorescence from the topically administeredfluorescently labelled ophthalmic formulation.

BRIEF DESCRIPTION OF THE DRAWINGS

The advantages and features of the present invention will appear fromthe following description when considered in conjunction with theaccompanying drawings in which:

FIG. 1 is a schematic of the apparatus in accordance with the presentinvention generally showing a support for a subject enablingillumination of an eye with a light source to cause fluorescence of afluorescent formulation disposed on the surface of an eye along with areceiver for measuring the fluorescence emitted from the eye;

FIG. 2 is a schematic representation of a receiver of fluorescent lightfrom a subject eye depicting a lens arrangement therein;

FIG. 3 is a plot showing the effect of varying viscosity ofhydroxypropylmethyl cellulose (HPMC) and polyvinyl alcohol (PVA) onfluorescein retention in the PTF; and

FIGS. 4-5 are plots showing the measurement of retention times ofophthalmic formulations in rabbits for up to 175 minutes enabled by thefluorescent macromolecules of the present invention.

DETAILED DESCRIPTION OF THE DRAWINGS

Turning now to FIG. 1, there is shown apparatus 10 in accordance withthe present invention which generally includes a base 12 and a subjectsupport assembly 14 attached thereto which provides means for supportinga subject and enabling light access to the subject's eye 16.

It should be appreciated that the support assembly 14 may be of anyconventional type and include members 18 conventionally adapted for thesupport of either a human or animal head and include a chin support 20or the like. In the case of a human subject, the support assembly 14 mayinclude a forehead support 22 and a post 24 for supporting the chinsupport 20 at a proper elevation.

A light projection assembly 30 includes a light source 32 and lenses 34along with a mirror 36 which in combination provide means forilluminating the subject's eye 16 with light of a selected frequencyrange in order to cause fluorescence of a fluorescent formulationtopically administered to the surface of the subject's eye. Thetransmission characteristics of the filter, of course, are dependentupon the excitation frequency of the fluorescent compound and, as anexample, for use with the fluorescent compounds hereinafter discussed,an appropriate excitation interference filter, such as a 490 nm centerwavelength with a 10 nm bandpass that was custom made by Omega Opticalof Brattleboro, Vt. A standard optical slit lamp such as a Nikkon FS IIslit lamp may be modified with the appropriate filters 34.

It should be appreciated that the filters 34 should be carefullyselected in order to eliminate stray excitation light which cansignificantly limit the sensitivity of the instrumentation. Asillustrated in FIGS. 1 and 2, the excitation light 42 is directed to thesubject's eye 16 for excitation of the fluorescently labelled ophthalmicformulation topically administered to the subject's eye 16. Uponexcitation, fluorescence occurs whereupon unpolarized light of adifferent wavelength is emitted from the ophthalmic formulation withsuch light 42 being directed through the mirror 36 into a viewingassembly 50 which includes an arm 52, upstanding from the base 12, whichis movable by means of a control lever 54 to adjust the emitted light 42into an objective system 56 supported by a fixture 58 connected to thearm 52. The objective system 56 is adapted to support: emission filters60, such as a 530 nm center wavelength with a 20 nm bandpass that wascustom made by Omega Optical. A photomultiplier tube 64 such as OrielCorporation, Stratford, Conn., Model No. 77349 may be attached to ahousing 66 of the objective system.

In turn, the photomultiplier tube 64 is interconnected with aconventional detector 66 such as an Oriel Model No. 7070 which may beconnected to a recorder 72 in a conventional manner. In combination, theobjective system 56, lenses 60, photomultiplier tube 64, detector 70,and recorder 72 provide means for measuring the fluorescent light fromthe thin precorneal tear film (not shown) and the filters 60 furtherprovide means for preventing measurable light of non-fluorescent lightfrequency from reaching the photomultiplier tube 64.

Ophthalmic formulations in accordance with the present invention formeasuring ophthalmic formulation retention in an eye may include apharmaceutical agent in an aqueous vehicle for an eye and a fluorescentmacromolecule having a molecular weight greater than about 2,000 Daltonsthat is dissolved in the vehicle. A large number of macromolecules aresuitable for combination with the pharmaceutical agent and includeFITC-Dextran, TRITC-Dextran, Phycobiliproteins and FITC-labelledsynthetic polymers or proteins. These polymers are available fromcommercial suppliers such as Molecular Probes, Eugene, Oreg.

The fluorescently labelled solution, when topically administered to theeye 16, is effectively spread evenly over the surface of the eye by theblinking mechanism. This fluorescent film is gradually eliminated fromthe eye through blinking and tear secretions. Since the molecular weightof the tracer is sufficiently large, diffusion of the tracer into oculartissue is completely eliminated. Hence, the. fluorescent label islocalized in the pre-corneal tear film which provides for superiorspatial resolution of fluorescence measurements.

Thus, the system, which includes the ophthalmic formulation and theapparatus hereinbefore described, is able to obtain a very accuratequantitative measurement of the ophthalmic formulation that resides onthe surface of an eye and the rate at which it is removed over time. Theremoval of the ophthalmic formulation is attributed primarily to normalblinking and lacrimation of the eye and not diffusion of the fluorescentlabel from the thin film on the eye 16. It should be appreciated thatthe fluorescent macromolecule has a molecular weight of greater than2,000 Daltons and preferably as much as 2,000,000 Daltons such asphycobiliproteins. Alternatively, the fluorescent macromolecule may be acombination of a fluorescent compound such as fluorescein isothiocyanatehaving a molecular weight of less than about 2,000 Daltons bonded to acompound such as Dextran having a molecular weight of 70,000 Daltons sothat the combined weight of the molecule, which is preferably covalentlybonded, is greater than 2,000 Daltons. Not only does the large size ofthe tracer prevent diffusion into the ocular tissue, but the tracermacromolecule will be entangled with any other macromolecules in theophthalmic formulation in order to prevent preferential diffusion of thetracer out of the tear film.

Heretofore measurements of retention time have been limited in duration,in part, due to the diffusion of low molecular weight tracers from thePTF. As shown in FIG. 3, as reported by Benedetto, Shah and Kaufman inInvestigative Ophthalmology, December 1975, if the low molecular weighttracer sodium fluorescein is used, then unless the viscosity of thevehicle is greater than about 20 cp, the retention times are less than10 minutes. As hereinabove pointed out, greater retention time can oftenbe achieved with higher viscosity formulations, but if the viscosity istoo large, then it is often accompanied by blurred vision and eyeirritation.

As shown in FIGS. 4-5 for New Zealand albino rabbits, the apparatus andmethod of the present invention are suitable for retention timemeasurement of up to 175 minutes by increasing the viscosity of theophthalmic formulation. Formulations A-B of FIG. 4 were made with 0.1%,by weight, FITC-Dextran (70,000 Daltons) and had viscosities of 5,000 cpand 300 cp, respectively, which are unaffected by the addition of theFITC Dextran. Comparing the retention times shown in FIGS. 3 and 4, itcan be seen that the macromolecule tracer of the present invention doesnot require a high viscosity formulation or vehicle in order to maintainits presence in the PTF, thus providing a reliable long term measurementof retention time through detection of emitted fluorescence.

Hence, the method of the present invention is ideally suited for thequantitative determination of retention times for ocular formulationsbecause the tracer does not preferentially diffuse out of theformulation and therefore does not give erroneous measurements.

FIG. 5 illustrates the usefulness of the present invention whencomparing the performance of various formulations. Comparison of curve A(viscosity equals 1,000 cp) with curve B (viscosity equals 10 cp), acommercially available artificial tear, shows that the higher viscosityformulation is definitely retained longer in the eye.

As hereinabove described, a fluorescent macromolecule suitable for usewith the apparatus and formulations of the present invention may includecommercially available fluorescent tracers having a molecular weightgreater than about 2,000 Daltons. However, a fluorescent molecule inaccordance with the present invention may also be produced by covalentlyattaching a fluorescent compound having a molecular weight of less thanabout 2,000 Daltons to a compound having a molecular weight sufficientto produce a macromolecule with a molecular weight greater than 2,000Daltons. For example, ##STR1##

REACTION A

Dissolve 20 mg Dextran in water adjusted to pH 11 with 0.2M NaOH.

Dissolve 10 mg cyanogen bromide in 0.2 ml water.

Mix Dextran and cyanogen bromide solutions together and maintain pH 11for 5 minutes.

Desalt activated Dextran using 1×20 cm column of Sephadex G-50 in 0.2Msodium borate at pH 8.0.

REACTION B

Pool Dextran-containing fractions.

Immediately react pooled Dextran samples with 2 mg of fluorescein (R)amine.

Let mixture react for 12-18 hours.

Separate fluorescein-Dextran from unreacted fluorescein amine usingSephadex G-50 1×20 cm column equilibrated in phosphate buffered saline.

From: Glabe, C. G.; Harty, P. K.; and Rosen S. D., "Preparation andProperties of Fluorescent Polysaccharides," Analytical Biochemistry, 130(1983) 287-294.

While fluorescent macromolecules having a molecular weight of less than2,000 Daltons may be useful, it has been found that diffusion of themacromolecule into ocular tissues is minimal at molecular weightsgreater than about 2,000 Daltons which enables the measurement ofretention time as long as the ophthalmic formulation is retained in theprecorneal region.

Separately and in combination with the hereinabove-described apparatus10, a method for measuring ophthalmic formulation retention on thesurface of an eye includes the steps of dissolving a fluorescentmacromolecule, such as FITC-Dextran, in an ophthalmic formulation toform a fluorescently labelled formulation having a concentration of0.1%, by weight of said fluorescent molecule. Thereafter, thefluorescently labelled formulation is topically administered to the eye16 with the fluorescently labelled formulation forming a thin film onthe eye surface. The light source 30 is used to illuminate the eye asthe fluorescently labelled formulation contained in the thin film iseliminated from the eye by normal blinking and lacrimation and theobjective system 56, with multiplier tube 64 and detector 70 areutilized for measuring the fluorescence from the thin film as a functionof time.

In the case where the macromolecule is not fluorescent, the method ofthe present invention may further comprise the step of covalentlyattaching a fluorescent compound having a molecular weight of less thanabout 2,000 Daltons to a compound to form the fluorescent moleculehaving a molecular weight of greater than 2,000 Daltons, as hereinabovedescribed.

The apparatus 10 in combination with a fluorescent macromolecule such asFITC-Dextran has provided a superior method for obtaining accuratequantitative measurements of precorneal retention times for ocularformulations. Its usefulness lies in the comparison of newly formulatedocular formulations with commercially available products or in theprocess of screening new formulations. Typical results of suchcomparison measurements are shown in FIG. 4 which is illustrative of thefact that the use of a fluorescently labelled macromolecule can measureretention times of up to 175 minutes.

In this procedure, the samples to be tested are prepared with anappropriate amount of fluorescent tracer material and instilled into thecul-de-sac of the animal or human subject. A baseline measurement ismade prior to sample installation by adjusting the sensitivity of thedetector system to the appropriate level expected in the experiment andfocussing the beam of the slit lamp on the precorneal tear film of thesubject.

The signal intensity of the baseline reading can then be subtractedusing instrumental controls in a conventional manner. The lightintensity and beam width and height are also set to prescribed values.Following the adjustments for the baseline, a sample is instilled in theeye and immediately after the sample has been evenly spread over thesurface of the eye with several quick blinks, a time zero measurement istaken. This measurement is made by quickly focussing the beam of theslit lamp on the precorneal tear film and reporting the photomultipliersignal on the printout from the recorder 72. This procedure can berepeated as often as required until the signal has returned to thebaseline indicating all of the sample has been eliminated from the eye.

Although there has been hereinabove described a specific arrangement andmethod for measuring ophthalmic formulation retention on the surface ofan eye in accordance with the present invention, for the purpose ofillustrating the manner in which the invention may be used to advantage,it should be appreciated that the invention is not limited thereto.Accordingly, any and all modifications, variations, or equivalentarrangements which may occur to those skilled in the art, should beconsidered to be within the scope of the present invention as defined inthe appended claims.

What is claimed is:
 1. A method for measuring ophthalmic formulation retention on a surface of an eye comprising the steps of:dissolving a fluorescent tracer in an ophthalmic formulation to form a fluorescently labelled formulation; topically administering the fluorescently labelled formulation to an eye, the topically administered formulation forming a thin film on an eye surface; allowing the fluorescently labelled formulation to be eliminated from the eye by normal blinking and lacrimation; preventing diffusion of the fluorescent tracer from the thin film and into ocular tissue by selecting the fluorescent tracer from a group consisting of fluorescent compounds having a molecular weight greater than 2,000 Daltons and fluorescent macromolecules comprising a fluorescent compound having a molecular weight less than 2,000 Daltons that is chemically attached to a non-fluorescent compound having a molecular weight sufficient to cause the fluorescent macromolecules to have a molecular weight greater than 2,000 Daltons; illuminating the eye to cause fluorescence of the fluorescently labelled formulation as the thin film is eliminated from the eye by normal blinking and lacrimation; and measuring fluorescence from the thin film.
 2. A method for measuring ophthalmic formulation retention on a surface of an eye comprising the steps of:topically administering a fluorescently labelled formulation to an eye, the fluorescently labelled formulation forming a thin film on an eye surface, said fluorescently labelled formulation comprising a fluorescent tracer and a pharmaceutical agent for the eye; allowing the fluorescently labelled formulation to be eliminated from the eye by normal blinking and lacrimation; preventing diffusion of the fluorescent tracer from the thin film and into ocular tissue by selecting the fluorescent tracer from a group consisting of (1) fluorescent compounds having a molecular weight greater than 2,000 Daltons and (2) fluorescent macromolecules comprising a fluorescent compound having a molecular weight less than 2,000 Daltons and a non-fluorescent compound having a molecular weight sufficient to cause the fluorescent macromolecules to have a molecular weight greater than 2,000 Daltons; illuminating the eye to cause fluorescence of the fluorescently labelled formulation as the thin film is eliminated from the eye by normal blinking and lacrimation; and measuring fluorescence from the thin film. 